Project Summary/Abstract Nearly one-third of the world population is infected with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). This reservoir contributes towards an increasing incidence of TB, with about 11 million new cases and 1.8 million deaths every year. The profound success of Mtb in causing disease depends on its ability to effectively utilize the host's lipid metabolism, including the sphingolipid biosynthesis pathway, to subvert the immune system. The granuloma, the pathological hallmark of TB infection, is characterized by the presence of lipid-loaded immune cells such as foamy macrophages that have a significantly reduced capability in controlling bacterial growth. Although Mtb is known for modulating lipid metabolism, and in particular the sphingolipid pathways during infection, there is no systematic understanding of how infective bacteria alter the host lipidome. We recently found that sphingolipids, a distinct class of lipids with a sphingoid base backbone that are enriched in cellular membranes, are essential for entry and killing of Mtb. We plan to extend our investigation to define the role of each sphingolipid repertoire in Mtb pathogenesis. The main goal of the proposed work is to systematically perturb the key sphingolipid biosynthetic pathways of the host to uncover their function in Mtb pathogenesis and antimicrobial cellular processes such as the inflammasome. For this purpose, individual knockout macrophage cell lines that lack key genes involved in the biosynthesis of sphingolipids will be generated using CRISPR/Cas9-technology. We will use multifunctional sphingolipid precursor analogs to define the flux, localization and the interactome of sphingolipids during infection in time- and space-dependent manner. Furthermore, we will study the significance of sphingolipids in the inflammasome, an innate immune process that is important in controlling Mtb infection. Understanding these pathways or processes essential for the pathogenesis of Mtb is crucial, as they represent potential targets for new therapeutics.